Blood transfusion is a common medical procedure that may be indicated for treatment of hemorrhage, hypoplastic anemia, hemolytic anemia, and other conditions. Although blood transfusion is a life-saving procedure, it carries with it the danger of an immune-mediated reaction by the transfusion recipient against the foreign (donor) cells. For example, if red blood cells (RBCs) are transfused into a recipient who mounts an immune response against the cells, an immune transfusion reaction may occur. The symptoms of a transfusion reaction can include malaise, fever, shock and renal failure, and will depend, in part, on whether whole blood or a blood component is transfused and on the particular antigen to which the immune response is mounted.
Because of the possibility of a transfusion reaction, it is routine for bloodbank practitioners to take steps to ensure that transfusion of the donor cells into a recipient results in a minimal immune response. This is done by determining the ABO and Rh types of a prospective transfusion recipient, and testing the recipient's serum for compatibility with the donor's red blood cells (e.g, by cross-matching or compatibility testing) prior to transfusion. In blood banking practice this screening usually begins with a triad of routine tests: an ABO test, a direct antiglobulin (Coombs) test, and an "antibody screening test." The ABO test is used to determine blood type. The direct Coombs test uses anti-IgG to agglutinate a patient's RBCs and, if positive, indicates that IgG are present on the surface of the patient's RBCs and suggests that an autoimmune response has been raised against these cells. The antibody screening test is used to detect the presence of an unexpected antibody in a patient's serum using reagent RBCs of known phenotype. If the antibody screen indicates that an alloantibody is present in the sample, another test such as a "compatibility test" (also known as a "panel test") is carried out to identify the classes of clinically significant alloantibodies, if any, in the recipient's serum. Panel tests consist of a series of Group O reagent RBC samples with known blood group phenotypes. The recipient's serum is combined with reagent RBCs of each phenotype, and the pattern of binding allows determination of what alloantibodies are present in the recipient's serum. For example, serum containing anti-Fy.sup.a alloantibodies will react with RBCs expressing the Fy.sup.a (Duffy) antigen but would not necessarily bind RBCs displaying other antigens. The term "antiglobulin test" is used to describe tests that can be used to identify alloantibodies. Antiglobulin tests are described in the American Association of Blood Banks (AABB) Technical Manual 11th Edition (1993) Bethesda, Md., which is incorporated herein by reference, at, e.g., pages 175-187. Indirect antiglobulin tests include antibody detection, antibody identification, and compatibility testing. When an indirect antiglobulin test is performed on a donor unit in preparation for infusion, it is referred to as compatibility testing. When the test is done on reagent red cells it is referred to as antibody identification or antibody screening.
Identification of alloantibodies in the recipient's serum by cross-matching or panel testing is important because some alloantibodies will react with, and rapidly destroy, transfused cells displaying the corresponding antigen. This results in a hemolytic transfusion reaction with potentially serious consequences for the recipient. Thus, whenever possible, patients with clinically significant alloantibodies should receive blood that has been found to lack the corresponding antigen. Alloantibodies are sometimes referred to as "unexpected alloantibodies" to distinguish them from anti-A or anti-B antibodies.
In some cases the antibody screen and/or panel test indicate that the recipient's serum contains antibodies that react with RBCs generally. When this result is accompanied by a positive Coombs test, a conclusion is made that the recipient's serum contains antibodies, referred to as warm autoantibodies, that react with antigens present on all RBCs. The name "warm autoantibodies" refers to the fact that these antibodies react optimally with RBCs at 37.degree. C. (in contrast to the cold agglutinins, which attach to red cells only when the temperature is below 37.degree. C. and dissociate from the cells when the temperature rises above 37.degree. C.) It has generally been believed that warm autoantibodies are primarily specific for Rh antigen and other protein antigens on RBCs. Warm autoantibodies are associated with conditions such as "warm antibody autoimmune hemolytic anemia;" however, not all patients with warm autoantibodies in their serum exhibit signs of anemia.
Because warm autoantibodies bind RBCs indiscriminately they mask the binding by alloantibodies to certain classes of RBCs in a panel test, making it impossible to determine what alloantibodies are present in a serum sample. For this reason, in order to detect alloantibodies it is necessary to first remove warm autoantibodies from a sample of the recipient's serum. According to current blood banking practice, warm autoantibodies are removed from a sample by a process such as autologous adsorption. In this procedure, red blood cells are obtained from the recipient and treated (for example, using chloroquine diphosphate or DTT with cysteine-activated papain) in order to elute autoantibody bound to them. The treated cells are then used to adsorb patient sera to remove warm autoantibody (e.g., by combining the enzyme-treated RBCs with an equal volume of serum, incubating for 60 minutes at 37.degree. C., and separating the cells from the adsorbed serum by centrifugation). The adsorbed serum is then used in a panel test against antibody screening cells. If all the cells in the panel remain positive, the autoadsorption procedure is repeated. It typically takes about 1.5 hours to complete one round of cell preparation and adsorption, and three to four rounds of absorption may be required to ensure removal of a sufficient amount of the warm autoantibody. Thus, it typically takes 4-6 hours to remove warm autoantibody from a serum sample. An alternative adsorption protocol in which allogenic RBCs are used for adsorption is employed when the patient has been recently transfused or when insufficient rbcs are available for autoadsorption.
The techniques available for removing warm autoantibodies from serum are time-consuming, tedious, and may require large amounts of cells and sera. Faster and more convenient methods are desirable and would be of great benefit to patients and health workers. In accordance with this need, the present invention provides new methods for antiglobulin testing that use methods other than autoadsorption for removing warm autoantibodies from serum.